Christian Slomianny

Bcl-2-dependent modulation of Ca2+ homeostasis and store-operated channels in prostate cancer cells

Antiapoptotic oncoprotein Bcl-2 has extramitochondrial actions due to its localization on the endoplasmic reticulum (ER); however, the specific mechanisms of such actions remain unclear. Here we show that Bcl-2 overexpression in LNCaP prostate cancer epithelial cells results in downregulation of store-operated Ca(2+) current by decreasing the number of functional channels and inhibiting ER Ca(2+) uptake through a reduction in the expression of calreticulin and SERCA2b, two key proteins controlling ER Ca(2+) content. Furthermore, we demonstrate that Ca(2+) store depletion by itself is not sufficient to induce apoptosis in Bcl-2 overexpressing cells, and that sustained Ca(2+) entry via activated store-operated channels (SOCs) is required as well. Our data therefore suggest the pivotal role of SOCs in apoptosis and cancer progression.

Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1)

Intestinal epithelium has the capacity to self-renew and generate differentiated cells through the existence of two types of epithelial stem cells: active crypt base columnar cells (CBCs) and quiescent +4 cells. The behaviors of these cells are regulated both by intrinsic programs and by extrinsic signals sent by neighboring cells, which define the niche. It is clear that the β-catenin pathway acts as an essential intrinsic signal for the maintenance and proliferation of CBC, and it was recently proposed that Paneth cells provide a crucial niche by secreting Wingless/Int (Wnt) ligands. Here, we examined the effect of disrupting the intestinal stem cell niche by inducible deletion of the transcription factor Math1 (Atoh1), an essential driver of secretory cell differentiation. We found that complete loss of Paneth cells attributable to Math1 deficiency did not perturb the crypt architecture and allowed the maintenance and proliferation of CBCs. Indeed, Math1-deficient crypt cells tolerated in vivo Paneth cell loss and maintained active β-catenin signaling but could not grow ex vivo without exogenous Wnt, implying that, in vivo, underlying mucosal cells act as potential niche. Upon irradiation, Math1-deficient crypt cells regenerated and CBCs continued cycling. Finally, CBC stem cells deficient in adenomatous polyposis coli (Apc) and Math1 were able to promote intestinal tumorigenesis. We conclude that in vivo, Math1-deficient crypts counteract the absence of Paneth cell-derived Wnts and prevent CBC stem cell exhaustion.

The antimalarial ferroquine: role of the metal and intramolecular hydrogen bond in activity and resistance.

Inhibition of hemozoin biocrystallization is considered the main mechanism of action of 4-aminoquinoline antimalarials including chloroquine (CQ) but cannot fully explain the activity of ferroquine (FQ) which has been related to redox properties and intramolecular hydrogen bonding. Analogues of FQ, methylferroquine (Me-FQ), ruthenoquine (RQ), and methylruthenoquine (Me-RQ), were prepared. Combination of physicochemical and molecular modeling methods showed that FQ and RQ favor intramolecular hydrogen bonding between the 4-aminoquinoline NH group and the terminal amino group in the absence of water, suggesting that this structure may enhance its passage through the membrane. This was further supported by the use of Me-FQ and Me-RQ where the intramolecular hydrogen bond cannot be formed. Docking studies suggest that FQ can interact specifically with the {0,0,1} and {1,0,0} faces of hemozoin, blocking crystal growth. With respect to the structure-activity relationship, the antimalarial activity on 15 different P. falciparum strains showed that the activity of FQ and RQ were correlated with each other but not with CQ, confirming lack of cross resistance. Conversely, Me-FQ and Me-RQ showed significant cross-resistance with CQ. Mutations or copy number of pfcrt, pfmrp, pfmdr1, pfmdr2, or pfnhe-1 did not exhibit significant correlations with the IC(50) of FQ or RQ. We next showed that FQ and Me-FQ were able to generate hydroxyl radicals, whereas RQ and me-RQ did not. Ultrastructural studies revealed that FQ and Me-FQ but not RQ or Me-RQ break down the parasite digestive vacuole membrane, which could be related to the ability of the former to generate hydroxyl radicals.

Role of Cationic Channel TRPV2 in Promoting Prostate Cancer Migration and Progression to Androgen Resistance

Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA-mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa.

The Protozoan Parasite Toxoplasma gondii Expresses Two Functional Plant-like Glycolytic Enzymes IMPLICATIONS FOR EVOLUTIONARY ORIGIN OF APICOMPLEXANS

The recent discovery of a vestigial, nonphotosynthetic plastid (“apicoplast”) in the Apicomplexa has considerably modified our perception of the evolutionary origin of these parasites. Phylogenetic analysis and the presence of four surrounding membranes of the apicoplast provide important support for the hypothesis that apicomplexans have acquired their apicoplast by secondary endosymbiosis, probably from a green alga. This suggests that genes encoding predicted homologs of proteins of green algae or related photosynthetic lineages could have entered the nucleus of apicomplexan parasites by transfer from the ancestor harboring the apicoplast. We describe here complementary DNAs encoding two Toxoplasma gondii glycolytic enzymes, glucose-6-phosphate isomerase (G6-PI) and enolase, which have considerable identities with land plant counterparts. Both cDNAs of T. gondii complementEscherichia coli mutants lacking G6-PI and enolase genes and lead to the expression of active enzymes. In the drug untreatable encysted bradyzoites of T. gondii, G6-PI and enolase genes are overexpressed or exclusively expressed at both transcriptional and protein levels. Moreover, three-dimensional models and protein phylogeny confirmed that G6-PIs and enolases of T. gondii,Plasmodium falciparum, and land plants are closely related. Because these glycolytic enzymes are plant homologs, which differ from those of animals, they will be useful to trace the evolutionary origin of Apicomplexa and might offer novel chemotherapeutic targets in diseases caused by apicomplexan parasites.

Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt

The serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: α -actinin, valosin-containing protein, inhibitor κB kinase, mortalin, tubulin β, cytokeratin 8, actin, 14-3-3σ, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt co-immunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling.

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line.

Prostate smooth muscle cells predominantly express α1‐adrenoceptors (α1‐AR). α1‐AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca2+ entry pathways associated with the activation of α1‐AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor‐operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura‐2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous α1A‐ARs, α‐agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca2+ influx which depended on the extracellular Ca2+ and PLC activation but was independent of PKC activation. Thus, we have tested two membrane‐permeable analogues of diacylglycerol (DAG), oleoyl‐acyl‐sn‐glycerol (OAG) and 1,2‐dioctanoyl‐sn‐glycerol (DOG). They initiated Ca2+ influx whose properties were similar to those induced by the α‐agonists. Sensitivity to 2‐aminoethyl diphenylborate (2‐APB), SKF‐96365 and flufenamate implies that Ca2+‐permeable channels mediated both α‐agonist‐ and OAG‐evoked Ca2+ influx. Following the sarcoplasmic reticulum (SR) Ca2+ store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca2+ influx. However, OAG failed to enhance Ca2+ influx when added in the presence of an α‐agonist. RT‐PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both α‐agonist‐ and OAG‐induced Ca2+ influx required TRPC3 and TRPC6, whereas the Tg‐activated (“capacitative”) Ca2+ entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor‐ and store‐operated Ca2+ entry pathways.

Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78

The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca2+ homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca2+ homeostasis, and the ER Ca2+ store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca2+ leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca2+ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca2+ leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca2+ permeability and subsequent ER stress can lead to development of a novel therapeutic approach.—Hammadi, M., Oulidi, A., Gackière, F., Katsogiannou, M., Slomianny, C., Roudbaraki, M., Dewailly, E., Delcourt, P., Lepage, G., Lotteau, S., Ducreux, S., Prevarskaya, N., Van Coppenolle, F. Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J. 27, 1600–1609 (2013). www.fasebj.org

Oleuropein and derivatives from olives as Tau aggregation inhibitors.

Tau isoforms constitute a family of microtubule-associated proteins that are mainly expressed in neurons of the central nervous system. They promote the assembly of tubulin monomers into microtubules and modulate their stability, thus playing a key structural role in the distal portion of axons. In Alzheimers disease and related tauopathies, Tau aggregation into fibrillary tangles contributes to intraneuronal and glial lesions. We report herein the ability of three natural phenolic derivatives obtained from olives and derived food products to prevent such Tau fibrillization in vitro, namely hydroxytyrosol, oleuropein, and oleuropein aglycone. The latter was found to be more active than the reference Tau aggregation inhibitor methylene blue on both wild-type and P301L Tau proteins, inhibiting fibrillization at low micromolar concentrations. These findings might provide further experimental support for the beneficial nutritional properties of olives and olive oil as well as a chemical scaffold for the development of new drugs aiming at neurodegenerative tauopathies.

A genetic study of the role of the Wnt/β-catenin signalling in Paneth cell differentiation

Wnt/beta-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/beta-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/beta-catenin pathway and mice with a hypomorphic beta-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/beta-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/beta-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in beta-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of beta-catenin activation is required to determine Paneth cell fate and that fine tuning of beta-catenin signalling is critical for correct Paneth cell lineage.